This invention relates to an electrical oxygen probe for the measurement of dissolved oxygen concentrations in fluids and of oxygen partial pressure in the air, particularly an electrical oxygen probe using a membrane electrode.
In the past for the measurement of dissolved oxygen concentration, polarographic and galvanic probes using membrane electrode were proposed. The polarographic membrane probe is provided with noble metals for both anode and cathode and requires the application of an applied voltage for the measurement of electrolytic reductive current of oxygen. The galvanic membrane probe is provided with a noble metal for cathode and a base metal for anode. Since this probe is a cell itself, no external electricity is required and the current derived from the reduction of oxygen is measured.
The prior technologies such as the said polarographic membrane probe and the said galvanic membrane probe were not sufficient in the durability for the steam sterilization which is carried out at elevated temperatures and at high pressures. Furthermore, when some parts of the probe such as membrane are raptured, the raptured parts can not be replaced for new ones. In other words, the whole probe becomes useless and a new one is required.
In general, in order to control the conditions of culture of microorganism and fermentation, it is essential, as is well known, to measure the oxygen concentration of the fermentation medium in the fermentor. However, prior to initiating the fermentation, the steam sterilization is usually carried out at the temperatures of 100.degree. C.-120.degree. C. Therefore it is expected that a membrane probe applicable and durable under such conditions is proposed.
In these past years, several improvements of such probes were proposed, but are not sufficient in their function yet. The reasons are that such a conventional probe is formed by individual separate parts such as an anode, a cathode and an electrolytic cell, then the structure is complicated, and that most of the said parts are connected with each other or insulated with each other by adhesives and the likes, then the connecting sections are ruptured easily by repeated sterilization at elevated temperatures and cooling in the culture and the fermentation. Furthermore, conventional probes have not sufficient durability in their materials and structures. Therefore it is difficult for users to repair probes by themselves in case that some parts of probes are ruptured. That is, such conventional probes have disadvantages in economics and operability.
For example, an improved galvanic membrane probe was reported as the title "The Value and Use of Dissolved Oxygen Measurement in Deep Culture" in the Chemical Engineer, No. 258 February 1972, p 63-71.
The outline of the art is as follows;
The essential part of the probe consists of a tubular glass body. For culture vessels of working volume greater than half a liter the glass body may be enclosed in a metal sheath. A PTFE (Teflon) membrane, 0.002 in thick, is sealed to one end with adhesive and held in position with a sleeve of silicone tube. The circular disc, which the membrane presents, forms the detecting element of the probe. The other end of the tube carries (i) the terminal connection for the anode and cathode, (ii) a filling tube, and (iii) a vent tube. In immediate contact with the membrane is a silver cathode consisting of a flat spiral of silver wire. Behind the silver is an anode composed of a helical spiral of lead sheet. The electrolyte is a mixture of sodium and lead acetates and acetic acid.
The cell is not sealed. Instead the gas space above the electrolyte is vented into the head space above the culture liquid. Thus, regardless of the internal pressure in the culture vessel, and in distinction to the conditions which apply in a sealed cell, the membrane is never subjected to a differential pressure greater than that of the head of culture liquid. This pressure is applied externally so that the silver cathode supports the membrane physically. Thus damage due to bursting stresses, produced during steam sterilizing, has been eliminated.
Although lack of sealing will enable oxygen to diffuse into the cell through the surface of the electrolyte, if such diffusion does occur, surprisingly it is not manifested as a residual current. It has been proposed that the effect of such diffusion is overcome because the oxygen is consumed by reaction with the upper part of the lead anode.
The silver-lead cell was chosen because the residual current at zero oxygen tension is small.
The electrolyte is of special composition as follows:
______________________________________ Acetic acid 5.0 M Sodium acetate 0.5 M Lead acetate 0.1 M pH value 3.0 approx. ______________________________________
Heating the probe to a temperature above the atmospheric boiling point imposes the need to take precautions during the cooling process.
Although the probe is designed to withstand repeated steam sterilization it can be damaged if it is not steam sterilized properly.
A probe in a culture vessel which is steamed in situ is never at risk, whether the vessel is empty or charged with medium, if at the end of steam treatment and during cooling, sterile air is introduced into the vessel to maintain a minimum total pressure, P.sub.m, which never falls below atmospheric, and is always greater than the corresponding aqueous vapour pressure, P.sub.a, of the culture medium or of the electrolyte in the probe. Thus evaporation and boiling are prevented. This is a routine practice designed to prevent concentration of the culture medium and, after the culture has cooled to below 100.degree. C., to prevent the ingress of contaminated ambient air, and the risk of mechanical collapse of the vessel, due to formation of a vacuum.
There is no risk if a laboratory vessel is sterilised in an autoclave of modern design which has provision for adding air under pressure during the cooling cycle in the manner described above.
The risks associated with cooling in an atmosphere of steam alone will now be discussed. If during cooling steam pressure falls too rapidly so that P.sub.a &lt;P.sub.m then water will evaporate or the electrolyte may even boil or bump out of the probe. If enough electrolyte is ejected so that the lead anode is not immersed the probe will not work. If the loss of electrolyte is partial so that the lead protrudes above the surface then after a few weeks the anode will break at the electrolyte surface. Again the probe will not work. If this method is employed then a probe should never be sterilized unless it is immersed in a bulk of medium or water, the time to cool to 100.degree. C. should be adjusted so that it is never less than 15 to 30 min. and the autoclave air vent should never be opened until the pressure has fallen to less than atmospheric.
Significant evaporation of water is an inevitable feature of this method of cooling and so there will be a cumulative loss of water during successive sterilizations. This will shorten probe life and change calibration conditions. Thus it will be seen that reliable operation, whether by treatment in situ or in an autoclave, is only possible if an air supply is used to minimise evaporation.
Having prevented boiling during cooling, it will be found that, for an initial treatment, one hour's exposure is sufficient. In subsequent use providing that the probe is never put on open circuit, the treatment time dictated by the needs of medium sterilization will generally be satisfactory.
The probe can be used for 30 to 40 batch cultures at 0.21 atm oxygen tension, each involving sterilization of the probe, before the output of current deteriorates to an unacceptable level. It is found that deterioration was rapid after the 28th sterilization.
The method of internal balancing, by exposing the vent hole to the gas space above a culture, puts a probe at risk if the culture foams. Medium may then enter the vent and gain access to the electrolyte. This risk is real in the case of the glass bodied type. In the shrouded variety where the glass body is encapsulated in a steel pipe there is a large internal capacity for foam. If foaming does occur there is a little risk of foam diluting the electrolyte, by gaining access through the secondary vents in the glass body.
The fragile nature of the membrane requires that the magnitude and direction of application of any pressure difference to which it is exposed shall be controlled. As a general rule the pressure outside the probe should be greater than the internal pressure. The silver cathode then acts as a support for the membrane. This improves dimensional stability and contributes to consistency of calibration. Experience has shown that, to reduce the risk of membrane rupture, it is unwise to immerse a probe, vented to the airspace in the vessel, to a greater depth than 10 ft of medium. That is, the recommended safe working pressure difference is 5 lbf in.sup.2. It is also axiomatic that the pressure difference should be held at a constant value to maintain dimensional and hence calibrational stability.
The said membrane probe requires a big membrane which is fixed at the bottom of glass tube and the area of which is about 10 times big as that of the glass tube bottom. The excess parts of the membrane is put on the glass tube wall and tightened by a silicon sleeve. Finally, the membrane is protected by covering a socket of stainless steel tube.
Accordingly, in cases that (a) the membrane is ruptured or loosened, (b) the activity and the output is lowered because of the stain of the cathode surface, (c) the electrolyte is spoiled, (d) the electrolyte is boiled off and reduced in the steam sterilization, the membrane probe cannot withstand to be used and a new one is required.